Summary: Murine hybridoma cells used in the production of monoclonal antibodies (mAbs) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAbs intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by evaluation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In a previous report, (Brorson et al., Biotechnol Prog. 188-96, 2001) we demonstrated the utility of TaqMan fluorogenic 5'nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assays for measuring reverse transcriptase (RT) activity in laboratory-scale cell-culture samples and RT removal by laboratory-scale models of processing steps. In follow up work, we evaluated the specificity, accuracy, range, precision and robustness of TM-PERT for this purpose. We found that this assay detects RT activity contained in xenotropic murine leukemia virus (X-MuLV) and CHO cell type C particles and quantifies particle numbers comparably to other assays (e.g., transmission electron microscopy, viral sequence specific TaqMan). Cell culture-derived DNA polymerases appeared to contribute only modestly to the assay background. Background amplification from intracellular DNA polymerases or other nuclear enzymes is potentially problematic only in culture samples with significant cell lysis. TM-PERT was linear and precise between 10e7 and 10e13 pU/mL, establishing the assay range. The assay was robust in that test article storage conditions and DNA/protein content had little impact on assay performance. Thus, TM-PERT appears to be an acceptable assay to measure type C particles in rodent cell culture samples. This work was published in Brorson et al., Biologicals, 30:15-26, 2002. Cell culture process changes (e.g. changes in scale, medium formulation, operational conditions) and cell line changes are common during the development phase of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, CBER collaborated with Genentech, Inc., to measure retrovirus expression in scale down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e. master cell bank vs. working cell bank) and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log10). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g. pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log10). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log10). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product specific productivity or alter the metabolic state, but suggest that re-assessment of retrovirus expression after other process changes may be unnecessary. Our collaborative work with Genentech was published in Brorson et al., Biotech and Bioengineering 80:257, 2002. Certain commercial purification modules, such as nanofiltration and low pH inactivation, have been observed to reliably clear greater than 4 log10 of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log10 reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the contents of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In collaboration with Genentech, Inc., CBER defined "bracketed generic clearance" conditions, using a prospectively defined bracket/matrix approach, where low pH inactivation consistently achieves >4.6 log10 clearance of Xenotropic murine leukemia virus (X-MuLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low pH treatment was also investigated. This collaborative work with Genentech, Inc., is in press at Biotech and Bioengineering. A potential safety concern in biotechnology purification schemes that employ re-use of column media, often for large numbers chromatography runs, is loss of the virus removal capacity of the chromatographic purification operation over time. To define chromatography performance quality attributes that best predict retrovirus clearance during extended re-use of protein A media, DMA has collaborated with ONDC/CDER to cycle small-scale protein A columns 150 to 460 times using concentrates of murine hybridoma cell culture supernatants, standard low pH elution buffers and different cleaning solutions (6M urea, 6M guanidine, 100 mM NaOH or 500mM NaOH). Load, flow-through and eluate samples were taken periodically and assayed for reverse transcriptase (RT, an enzyme component of retroviruses) activity, bovine IgG (a component of the culture media), genomic DNA, leached protein A, and mouse IgG. Under all cleaning conditions tested, the log10 reduction value (LRV) of RT activity did not decrease and impurity co-elution did not increase during the 150 to 460 purification/cleaning cycles. In the two studies in which the columns were cleaned with NaOH, the chromatography performance quality attribute that best predicted the column media lifespan was column capacity, as measured by Ab step yield and breakthrough. In both studies, Ab capture decayed in a biphasic manner starting at cycle 200 (100mM NaOH) or cycle 50 (500mM NaOH). For media cycled 300+ times using 6M urea or 6M guanidine cleaning buffers, column performance, including RT activity LRV, was more stable, although small upward trends in Ab breakthrough were evident. In summary, our studies identify Ab step yield and breakthrough as performance quality attributes that decay prior to retrovirus LRV when protein A media is multiply-cycled. Thus, we propose that virus removal validation studies should be performed on new media only and these quality attributes can be monitored during protein A unit operations in lieu of performing virus removal validation studies with cycled protein A media. This collaborative work with ONDC/CDER has been submitted to the Journal of Chromatography. DMA is currently working with ONDC/CDER and others to expand the media decay studies to other resin types, including ion exchange resins.